The present invention relates to compositions and methods for treating pain. Particularly, the present invention relates to an agent comprising a neurotoxin, methods for making the agent and methods for treating pain using the agent.
xe2x80x9cFor all the happiness mankind can gain is not in pleasure, but rest from pain.xe2x80x9d John Dryden (1631-1700).
It is convenient to divide the human pain experience into two general categories, acute and chronic. Any noxious stimulus, for example extreme heat or sharp objects, may elicit an acute pain. The pain resulting from such a stimulus usually subsides in a relatively short period of time. Acute pain may also present itself in the course of any disease. However, such pain is also self-limited and subsides with time or adequate treatment.
Chronic pain is the second major category of pain experience. It can be defined as significant pain persisting for more than a few weeks for which there is no adequate therapy available to treat the underlying problem. Globally, there are countless numbers of people who presently are victims of chronic pain. For example, just in the United States alone, the National Institute of Health estimates that more than 90 million Americans suffer from chronic pain stemming from migraine headaches, back pain, arthritis, trauma, allodynia or catastrophic illness.
In general, the transduction of acute or chronic pain signals from the periphery to sensation itself is achieved by a multi-neuronal pathway and the information processing centers of the brain. The first nerve cells of the pathway involved in the transmission of sensory stimuli are called primary sensory afferents. The cell bodies for the primary sensory afferents from the head and some of the internal organs reside in various ganglia associated with the cranial nerves, particularly the trigeminal nuclei and the nucleus of the solitary tract. The cell bodies for the primary sensory afferents for the remainder of the body lie in the dorsal root ganglia of the spinal column. The primary sensory afferents and their processes have been classified histologically; the cell bodies fall into two classes: A-types are large (60-120 micrometer in diameter) while B-types are smaller (14-30 micrometer) and more numerous. Similarly the processes fall into two categories: C-fibers lack the myelin sheath that A-fibers possess. A-fibers can be further sub-divided into A beta-fibers, that are large diameters with well-developed myelin, and A delta-fibers, that are thinner with less well developed myelin. It is generally believed that A beta-fibers arise from A-type cell bodies and that A delta- and C-fibers arise from B-type cell bodies.
After the activation of the primary sensory afferents the next step in the transduction of sensory signals is the activation of the projection neurons, which carry the signal, via the spinothalamic tract, to higher parts of the central nervous system such as the thalamic nuclei. The cell bodies of these neurons (other than those related to the cranial nerves) are located in the dorsal horn of the spinal cord, This is also where the synapses between the primary afferents and the projection neurons are located. The dorsal horn is organized into a series of laminae that are stacked, with lamina I being most dorsal followed by lamina II, etc. The different classes of primary afferents make synapses in different laminae. For cutaneous primary afferents, C-fibers make synapses in laminae I and II, A delta-fibers in laminae I, II, and V, and A beta-fibers in laminae III, IV, and V. Deeper laminae (V-VII, X) are thought to be involved in the sensory pathways arriving from deeper tissues such as muscles and the viscera.
The predominant neurotransmitters at the synapses between primary afferents and projection neurons are substance P, glutamate, calcitonin-gene related peptide (CGRP) and neuropeptide Y. The efficiency of transmission of these synapses can be altered via descending pathways and by local interneurons in the spinal cord. These modulatory neurons release a number of mediators that are either inhibitory (e.g. opioid peptides, glycine, norepinephrine) or excitatory (e.g. nitric oxide, cholecystokinin, norepinephrine), to provide a mechanism for enhancing or reducing awareness of sensations.
Although the present available treatments for acute pain are usually manageable, the treatments for chronic pain are inadequate and disappointing. For example, it is known that intraspinal administration of opioids, such as morphine and fentanyl can alleviate pain. See e.g. Gianno, J., et al., Intrathecal Drug Therapy for Spasticity and Pain, Springer-Verlag (1996) (which publication is incorporated herein by reference in its entirety). However, these drugs used in intraspinal, or intrathecal, injections typically have only short lived antinociceptive effects. As a result, these drugs have to be frequently administered, such as by the aid of a pump for continuous infusion. For example, one frequently used pump is the SynchroMed(copyright) Infusion System, a programmable, implanted pump available from Medtronic, Inc., of Minneapolis, Minn. However, complications can arise due to the required surgical implantation procedure for the use of the pump and the known intrathecally administered drugs for pain, such as opioids, have the disadvantages of dependency and potential respiratory depression.
Longer acting analgesics are also known, for example, blocks by phenol injection. However, such treatments raise a considerable risk of irreversible functional impairment.
The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals referred to as botulism. The spores of Clostridium botulinum are found in soil and can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism. The effects of botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium botulinum culture or spores. The botulinum toxin can apparently pass unattenuated through the lining of the gut and attack peripheral motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.
Botulinum toxin type A is the most lethal natural biological agent known to man and has a very potent LD50. A specific dose of a toxin that would be lethal to 50% of the population of a certain species of animal is called an LD50. For example, the estimated LD50 of botulinum toxin type A (available from Allergan, Inc., of Irvine, Calif. as a purified neurotoxin complex under the trade name BOTOX(copyright)) in humans is about 150,000 picograms or about 3,000 units. Interestingly, on a molar basis, botulinum toxin type A is about 1.8 billion times more lethal than diphtheria toxin, about 600 million times more lethal than sodium cyanide, about 3.0 million times more lethal than cobra toxin and about 12 million times more lethal than cholera toxin. Singh, Critical Aspects of Bacterial Protein Toxins, pages 63-84 (chapter 4) of Natural Toxins II, edited by B. R. Singh et al., Plenum Press, New York (1996).
Seven immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C1, D, E, F and G, each of which is distinguished by neutralization with type-specific antibodies. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. For example, it has been determined that botulinum toxin type A is 500 times more potent, as measured by the rate of paralysis produced in the rat, than is botulinum toxin type B. Botulinum toxin apparently binds with high affinity to cholinergic motor neurons, is translocated into the neuron and blocks the release of acetylcholine.
Without wishing to limit the invention to any theory or mechanism of operation, it is believed that the molecular mechanism of toxin intoxication appears to be similar and involve at least three steps or stages, regardless of the serotype. Although, a potential molecular mechanism of toxin intoxication of botulinum toxin is discussed here, other toxins, for example, butyricum toxins, tetani toxins or variants thereof may have the same or substantially similar mechanisms. In the first step of the process, the toxin binds to the presynaptic membrane of the target neuron through a specific interaction between the heavy chain, H chain, and a cell surface receptor; the receptor is thought to be different for each type of botulinum toxin and for tetanus toxin. The carboxyl end segment of the H chain, HC, appears to be important for targeting of the toxin to the cell surface.
In the second step, the toxin crosses the plasma membrane of the poisoned cell. The toxin is first engulfed by the cell through receptor-mediated endocytosis, and an endosome containing the toxin is formed. The toxin then escapes the endosome into the cytoplasm of the cell. This step is thought to be mediated by the amino end segment of the H chain, HN, which triggers a conformational change of the toxin in response to a pH of about 5.5 or lower. Endosomes are known to possess a proton pump, which decreases intra-endosomal pH. The conformational shift exposes hydrophobic residues in the toxin, which permits the toxin to embed itself in the endosomal membrane. The toxin (or at a minimum the light chain) then translocates through the endosomal membrane into the cytoplasm of the cell.
The last step of the mechanism of botulinum toxin activity appears to involve reduction of the disulfide bond joining the heavy chain, H chain, and the light chain, L chain. The entire toxic activity of botulinum and tetanus toxins is contained in the L chain of the holotoxin; the L chain is a zinc (Zn++) endopeptidase, which selectively cleaves proteins essential for recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane. Tetanus neurotoxin, botulinum toxin /B/D/F, and /G cause degradation of synaptobrevin (also called vesicle-associated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the VAMP present at the cytoplasmic surface of the synaptic vesicle is removed as a result of any one of these cleavage events. Serotype A and E cleave SNAP-25. Serotype C1 was originally thought to cleave syntaxin, but was found to cleave syntaxin and SNAP-25. Each toxin specifically cleaves a different bond (except tetanus and type B, which cleave the same bond).
Botulinum toxins have been discovered to have relatively prolonged neurotoxic effects and, as such, have been adapted for use in the treatment of pain, particularly chronic pain, for example, Foster et al. in U.S. Pat. No. 5,989,545, the disclosure of which is incorporated in its entirety herein by reference.
However, most drugs presently being used for treating pain, for example chronic pain, are still inadequate. For example, one type of chronic pain is allodynia. Allodynia is a condition wherein normal non-noxious stimuli elicit pain. Presently known compounds may partially alleviate the conditions of allodynia, but at the same time eliminate the ability of a patient to sense any pain altogether, such as acute pain caused by a noxious stimulus. The ability to detect pain resulting from a noxious stimulus is importantxe2x80x94it allows for self-preservation. Also, many agents have undesired side effects, for example sedation, mood changes and/or hypotension. Finally, most such agents have a short therapeutic duration.
Therefore, there continues to be a need to have compounds which are selective and/or long acting for treating pain, preferably chronic pain.
In accordance with the present invention, an agent is featured comprising a therapeutic component and a targeting component, which selectively binds at either the alpha-2B or the alpha-2B and alpha-2C adrenergic receptor subtype(s) as compared to the alpha-2A adrenergic receptor subtype at the cell surface. Preferably, such cell is a neuron. In one embodiment, the agent further comprises a translocation component.
Further in accordance with the present invention, an agent according to this invention may be useful for treating pain, particularly chronic pain, in a mammal, including a human. Additionally, an agent according to this invention may be used to treat chronic pain, for example allodynia, without substantially affecting acute pain sensation or tactile sensation.
Still further in accordance with the present invention, the therapeutic component substantially interferes with the release of neurotransmitters from a cell or its processes. For example, in one embodiment, the therapeutic component comprises a light chain component, which may be able to inhibit the release of neurotransmitters from a cell. The light chain component may be a light chain or a fragment thereof of a Clostridial toxin such as a botulinum toxin type A, B, C1, D, E, F, G, a butyricum toxin, a tetani toxin or variants thereof. In another embodiment, the therapeutic component may be a neurotoxin, for example saporin, through inactivating cellular ribosome functions.
Still further in accordance with the invention, the targeting components may be molecules or amino acid components. Amino acid components include, for example, peptides, polypeptides, proteins, protein complexes, and antibodies, provided that these species selectively bind at the alpha-2B or alpha-2B/alpha-2C adrenergic receptor subtype(s). In one embodiment, the molecules may be imiloxan, ARC 239, prazosin or molecules represented by the formula: 
wherein Xxe2x80x2 is selected from the group consisting of R4xe2x80x94Cxe2x95x90Cxe2x80x94R5 and R4xe2x80x94C. A six membered carbon ring structure is formed when Xxe2x80x2 is R4xe2x80x94Cxe2x95x90Cxe2x80x94R5. A five membered carbon ring is formed when Xxe2x80x2 is R4xe2x80x94C. R1, R2, R3, R4 and R5 are each independently selected from the group consisting of F, Cl, Br, I, OR6 and H, wherein R6 is H or an alkyl, including a methyl, an ethyl or a propyl. In one embodiment, the amino acid component may be antibodies raised from an antigen component. The antigen component may include a second extracellular loop of an alpha-2B receptor, which may additionally be conjugated to a keyhole limpet hemocyanin. In one embodiment, the second extracellular loop comprises a peptide fragment comprising an amino acid sequence of KGDQGPQPRGRPQCKLNQE (SEQ ID#1). In another embodiment, the amino acid component may comprise a peptide, polypeptide, protein, protein complex or antibody, which is a variant of a wild type. For example, an amino acid component may be a mutated H chain of botulinum toxin type A which selectively binds to an alpha 2B receptor, as opposed to the wild type which has a higher affinity to motor neuron cell surface proteins. See Goeddel et al. U.S. Pat. No. 5,223,408, the disclosure of which is incorporated in its entirety herein by reference.
Still further in accordance with the present invention, the translocation component is able to facilitate the transfer of a therapeutic component, such as a light chain of a botulinum toxin type A, into the cytoplasm of the target cell. In one embodiment, the translocation component comprises a heavy chain component. The heavy chain component may include a heavy chain or a fragment thereof of a Clostridial toxin such as a botulinum toxin type A, B, C1, D, E, F, G, a butyricum toxin, a tetani toxin or variants thereof. The fragment of the heavy chain may include an amino end fragment of the heavy chain. In another embodiment, the heavy chain component may comprise at least a fragment of two different neurotoxins. For example, the heavy chain component may comprise an amino end fragment of heavy chain of a botulinum toxin type A, and a carboxyl end fragment of a heavy chain of botulinum toxin type B.
Still further in accordance with the invention, the therapeutic component, the translocation component and the targeting component are joined by one or more spacer component. For example, the therapeutic component may be joined to the translocation component through a spacer component, and the therapeutic component may be joined to the targeting component through a spacer component. In one embodiment, the spacer component comprises a moiety selected from the group consisting of a hydrocarbon, a polypeptide other than an immunoglobulin hinge region, and a proline-containing polypeptide identical or analogous to an immunoglobulin hinge region. In another embodiment, the therapeutic component may be joined to the translocation component through a spacer component, and the therapeutic component may be joined to the targeting component through a disulfide bridge.
Still further in accordance with the invention, there is provided a method for making an agent of the present invention comprising the step of producing a polypeptide from a gene, which encodes for at least one component of the agent, for example the therapeutic component, the translocation component and/or the targeting component.
Still further in accordance with the invention, there is provided a method for treating pain comprising the step of administering to a mammal, preferably a human, a therapeutically effective amount of an agent of the present invention. In one embodiment, the therapeutic component and the translocation component of the agent is found together in a botulinum toxin, for example botulinum toxin type A. An agent of the present invention may be administered intrathecally or intramuscularly or subcutaneously, for example at or near the location of pain.
Still further in accordance with the invention, the agent may be employed to treat chronic pain. More preferably, the agent may be employed to treat allodynia. Even more preferably the agent may be employed to treat allodynia without substantially affecting acute pain sensation or tactile sensation. Without wishing to limit the invention to any particular theory or mechanism of operation, it is believed that the selectivity of treating allodynia without affecting acute pain or tactile sensation, as described above, is due to the agent acting selectively on alpha 2B and/or alpha 2C receptors.